U.S. patent number 9,243,221 [Application Number 13/131,041] was granted by the patent office on 2016-01-26 for compositions and methods of functionally enhanced in vitro cell culture system.
This patent grant is currently assigned to THE GENERAL HOSPITAL CORPORATION, HUREL CORPORATION. The grantee listed for this patent is Robert Freedman, Yaakov Nahmias, Eric Novik, Martin Yarmush. Invention is credited to Robert Freedman, Yaakov Nahmias, Eric Novik, Martin Yarmush.
United States Patent |
9,243,221 |
Yarmush , et al. |
January 26, 2016 |
Compositions and methods of functionally enhanced in vitro cell
culture system
Abstract
Compositions and methods described herein provide a cell culture
system in which cells are in high metabolic states from the onset
of the culture. Combinations of various cell culture components
disclosed and employed herein allow cells to be in high metabolic
states useful for drug testing immediately after the start of cell
culture.
Inventors: |
Yarmush; Martin (Newton,
MA), Freedman; Robert (Beverly Hills, CA), Nahmias;
Yaakov (Plymouth, MN), Novik; Eric (Edison, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Yarmush; Martin
Freedman; Robert
Nahmias; Yaakov
Novik; Eric |
Newton
Beverly Hills
Plymouth
Edison |
MA
CA
MN
NJ |
US
US
US
US |
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|
Assignee: |
HUREL CORPORATION (Beverly
Hills, CA)
THE GENERAL HOSPITAL CORPORATION (Boston, MA)
|
Family
ID: |
42226369 |
Appl.
No.: |
13/131,041 |
Filed: |
November 24, 2009 |
PCT
Filed: |
November 24, 2009 |
PCT No.: |
PCT/US2009/065781 |
371(c)(1),(2),(4) Date: |
February 02, 2012 |
PCT
Pub. No.: |
WO2010/062911 |
PCT
Pub. Date: |
June 03, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120129207 A1 |
May 24, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61118362 |
Nov 26, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M
23/12 (20130101); C12N 5/0671 (20130101); C12M
35/08 (20130101); C12M 25/14 (20130101); C12M
23/16 (20130101); C12N 2500/02 (20130101); C12N
2513/00 (20130101); C12N 2500/90 (20130101) |
Current International
Class: |
C12N
5/071 (20100101); C12M 1/12 (20060101); C12M
1/32 (20060101); C12M 3/06 (20060101); C12M
1/42 (20060101); C12Q 1/02 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1548031 |
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Jun 2005 |
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EP |
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WO 2005063809 |
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Jul 2005 |
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WO |
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Other References
Schiff et al., Organ Culture of Adult Rat Colonic Mucosa on Fibrin
Foam, In Vitro, 1980, vol. 16, pp. 893-906. cited by examiner .
Hung et al., Continuous Perfusion Microfluidic Cell Culture Array
for High-Throughput Cell-Based Assays, Biotechnology and
Bioengineering , 2004, vol. 89, pp. 1-8. cited by examiner .
Nahmias et al., A novel formulation of oxygen-carrying matrix
enhances liver-specific function of cultured hepatocytes, The FASEB
Journal, 2006, vol. 20, pp. E1828-E1836. cited by examiner .
Evdokimova et al., Effects of bacterial endotoxin
(lipopolysaccharides) on survival and metabolism of cultured
precision-cut rat liver slices, Toxicology in Vitro, 2002, vol. 16,
pp. 47-54. cited by examiner .
Krause et al., Hepatocytmupported serum-free culture of rat liver
sinusoidal endothelial cells, Journal of Hepatology, 2000, vol. 32,
pp. 718-726. cited by examiner .
Corning Inc., Life Sciences, Reducing Serum Levels and Culture
Costs, 2005, pp. 1-4. cited by examiner .
Van de Bovenkamp et al., Precision-cut fibrotic rat liver slices as
a new model to test the effects of anti-fibrotic drugs in vitro,
Journal of Hepatology, 2006, vol. 45, pp. 696-703. cited by
examiner .
Van de Bovenkamp et al., Liver fibrosis in vitro: Cell culture
models and precision-cut liver slices, Toxicology in Vitro, 2007,
vol. 21, pp. 545-557. cited by examiner .
Boess et al., Gene Expression in Two Hepatic Cell Lines, Cultured
Primary Hepatocytes, and Liver Slices Compared to the in Vivo Liver
Gene Expression in Rats: Possible Implications for Toxicogenomics
Use of in Vitro Systems, Toxicological Sciences, 2003, vol. 73, pp.
386-402. cited by examiner .
International Search Report for PCT/US2009/065781, mailed Jul. 30,
2010. cited by applicant .
Written Opinion of the International Searching Authority for
PCT/US2009/065781, mailed 30, 2010. cited by applicant .
Kidambi, S. et al., "Oxygen-mediated enhancement of primary
hepatocyte metabolism, functional polarization, gene expression,
and drug clearance," PNAS, vol. 106, No. 37, pp. 15714-15719
(2009). cited by applicant .
Rotem, A. et al., "Oxygen Is a Factor Determining In Vitro Tissue
Assembly: Effects on Attachment and Spreading of Hepatocytes,"
Biotechnology and Bioengineering, vol. 43, pp. 654-660 (1994).
cited by applicant .
Suleiman S.A. et al., "The Effect of Oxygen Tension on Rat
Hepatocytes in Short-Term Culture," In Vitro Cellular &
Developmental Biology, vol. 23, No. 5, pp. 333-338 (1987). cited by
applicant .
Tilles, A. W., et al., "Effects of Oxygenation and Flow on the
Viability and Function of Rat Hepatocytes Cocultured in a
Microchannel Flat-Plate Bioreactor," Biotechnol Bioeng, vol. 73,
pp. 379-389 (2001). cited by applicant .
Yanagi, K. et al., "Improvement of Metabolic Performance of
Cultured Hepatocytes by High Oxygen Tension in the Atmosphere,"
Artificial Organs, vol. 25, No. 1, pp. 1-6 (2001). cited by
applicant.
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Primary Examiner: Humphrey; Louise W
Assistant Examiner: Keller; Christopher
Attorney, Agent or Firm: Law Office of Salvatore Arrigo and
Scott Lee, LLP
Government Interests
GOVERNMENT SUPPORT
This invention was made with Government support under Grant Numbers
EB002503 and DK080241 awarded by the National Institutes of Health.
The Government has certain rights in this invention.
Parent Case Text
CROSS-REFERENCE
This application is the U.S. National Stage of International
Application No. PCT/US2009/065781, filed Nov. 24, 2009, which
claims the benefit of U.S. Provisional Application No. 61/118,362,
filed Nov. 26, 2008, both of which are incorporated herein by
reference.
Claims
What is claimed is:
1. A cell culture system, comprising: a) a cell culture compartment
comprising a cell culture substrate and a cell culture medium
comprising no more than about 10% serum; b) a coculture of
metabolically active primary hepatocytes and at least one other
cell type, said coculture seeded onto the culture substrate and
cultured in the culture medium; and c) a gaseous composition in
contact with the culture medium and comprising at least about 90%
oxygen.
2. The cell culture system of claim 1, wherein the culture medium
comprises no more than about 5% serum.
3. The cell culture system of claim 1, wherein the culture medium
is serum free.
4. The cell culture system of claim 1, wherein the at least one
other cell type is a stromal cell type.
5. The cell culture system of claim 1, wherein the at least one
other cell type is a non-parenchymal cell type.
6. The cell culture system of claim 1, wherein the ratio of the
number of metabolically active primary hepatocytes to the number of
cells of the at least one other cell type in the coculture is from
about 1:10 to about 10:1.
7. The cell culture system of claim 1, wherein the ratio of the
number of metabolically active primary hepatocytes to the number of
cells of the at least one other cell type in the coculture is from
about 1:5 to about 5:1.
8. The cell culture system of claim 1, wherein the gaseous
composition in contact with the culture medium comprises at least
about 95% oxygen.
9. The cell culture system of claim 1, wherein the gaseous
composition in contact with the culture medium comprises at least
about 100% oxygen.
10. The cell culture system of claim 1, wherein the coculture of
metabolically active primary hepatocytes and the at least one other
cell type are seeded into the culture compartment and cultured in
the culture medium in contact with the gaseous composition for
about one day.
11. The cell culture system of claim 10, wherein following
culturing in the culture medium in contact with the gaseous
composition for about one day the level of albumin secretion by
hepatocytes in the coculture is at least about 4-fold higher than
the level of albumin secretion by hepatocytes in a control
coculture comprising a gaseous composition in contact with the
culture medium and comprising an atmospheric level of oxygen.
12. The cell culture system of claim 10, wherein the level of
albumin secretion by hepatocytes in the coculture is at least about
80 .mu.g/1.times.10.sup.6 cells /24 hrs.
13. The cell culture system of claim 10, wherein the level of
transcription of phase I and phase II enzymes in hepatocytes in the
coculture is comparable to the level of transcription of phase I
and phase II enzymes in freshly isolated hepatocytes.
14. The cell culture system of claim 10, wherein the CYP1A1/2
activity level of hepatocytes in the coculture is comparable to the
CYP1A1/2 activity level in freshly isolated hepatocytes.
15. A cell culture system made by a method comprising: a) seeding a
coculture of metabolically active primary hepatocytes and at least
one other cell type onto a culture substrate; and b) culturing the
seeded coculture in a culture medium comprising no more than about
10% serum; wherein during the seeding and/or culturing the culture
medium is in contact with a gaseous composition comprising at least
about 90% oxygen.
16. The cell culture system of claim 15, wherein the culturing is
for from about one to about three days.
17. The cell culture system of claim 15, wherein the culture medium
comprising no more than about 5% serum.
18. The cell culture system of claim 15, wherein the culture medium
is serum free.
19. The cell culture system of claim 15, wherein the gaseous
composition comprises at least about 95% oxygen.
20. The cell culture system of claim 15, wherein the gaseous
composition comprises at least about 100% oxygen.
21. A cell culture system, comprising: a) a cell culture
compartment comprising a cell culture substrate and a cell culture
medium comprising no more than about 10% serum; b) a coculture of
metabolically active primary hepatocytes and at least one other
cell type, said coculture seeded onto the culture substrate and
cultured in the culture medium, wherein during the seeding and/or
the first one to three days of culturing the culture medium is in
contact with a gaseous composition comprising at least about 90%
oxygen; and c) a gaseous composition in contact with the culture
medium and comprising at least about 90% oxygen; wherein the level
of albumin secretion by hepatocytes in the coculture is at least
about 4-fold higher than the level of albumin secretion by
hepatocytes in a control coculture comprising a gaseous composition
in contact with the culture medium and comprising an atmospheric
level of oxygen.
22. The cell culture system of claim 21, wherein the level of
albumin secretion by hepatocytes in the coculture is at least about
80 .mu.g/1.times.10.sup.6 cells /24 hrs.
23. The cell culture system of claim 21, wherein the level of
transcription of phase I and phase II enzymes in hepatocytes in the
coculture is comparable to the level of transcription of phase I
and phase II enzymes in freshly isolated hepatocytes.
24. The cell culture system of claim 21, wherein the CYP1A1/2
activity level of hepatocytes in the coculture is comparable to the
CYP1A1/2 activity level in freshly isolated hepatocytes.
Description
BACKGROUND OF THE INVENTION
The liver constitutes a central site in the absorption, binding,
distribution, metabolism, excretion, and toxicogenicity
(absorption, distribution, metabolism, excretion and toxicity,
"ADME-T") of xenogenous materials (i.e. materials foreign to the
body in their origination). When a xenobiotic entity, such as a
drug, pharmaceutical, or nutriceutical, enters a human body, it is
frequently cleared (i.e., metabolically disposed of) in the liver
by oxidation, reduction, hydroloysis, and/or hydration steps of
biochemical reaction. Of the over a dozen different cell types that
comprise the liver, the hepatocyte is the type primarily
responsible for playing the role of "clearing house" or
"biotransformation driver," metabolically disposing of xenogenous
material. In the liver, the hepatocyte is the cell type wherein a
family of enzymes named cytochrome P-450 or CYP450 are chiefly
expressed, along with other enzymes that also mediate the Phase I
as well as the Phase II metabolic disposition of drugs, and other
xenogenous materials. The various CYP450 isozymes collectively
comprise the most important group of metabolizing enzymes that
perform the role of clearing house. The field of study of how the
body disposes of xenobiotic entities is frequently called Drug
Metabolism and Pharmacokinetics, or DMPK. The term
"pharmacokinetics" is often used in contradistinction to the term
"pharmacodynamics." Pharmacodynamics signifies the impacts and
effects that a drug may biochemically exert upon a cell, an organ
or an entire animal; whereas pharmacokinetics signifies the
impacts, effects and ultimate disposition that a cell, organ or
entire animal may biochemically exert upon a xenogenous chemical
entity. In everyday language, pharmacodynamics comprises what the
drug does to the body, while pharmacokinetics comprises what the
body does to the drug. Toxicity, including hepatotoxicity, is a
major category of pharmacodynamic effect (drug efficacy being
another such major category); while drug absorption, metabolism,
distribution, and excretion comprise the major categories of
pharmacokinetic effects.
In addition to metabolic function, signaling interactions
constitute another important category of cellular function in the
liver and in other organs. Classes of proteins called chemokines or
cytokines, among others, frequently effectuate signaling
interactions. Modern biotechnology has led to the delineation of a
variety of molecular signaling pathways in the cell. These
signaling pathways not only have provided insights into the
mechanisms of a cell, but also have opened opportunities to
intervene with cellular processes or abnormalities. Antibodies,
vaccines and other forms of chemical entities have been utilized to
specifically promote, inhibit, induce, or reduce one or more
signaling pathways.
Many attributes of a molecular entity must be investigated and in
some cases chemically modified or improved in the course of
developing that molecular entity into a therapeutically efficacious
and safe compound that regulatory agencies approve for marketing
and clinical use. One challenge is investigating and if necessary
overcoming any toxicity that the drug candidate may directly or
indirectly induce. Another set of challenges is to understand and
in some circumstances to improve the pharmacokinetic properties of
the molecular entity. Studying and if possible improving the
efficacy of the molecular entity to achieve an intended biochemical
result comprises a third set of challenges to be addressed along
the path of discovering, developing, testing, and ultimately
receiving marketing approval for a new drug.
To address the kinds of challenges enumerated above, in vitro
cell-based assay systems are frequently utilized to simulate,
measure and/or predict various functional attributes (including
without limitation those elaborated in the paragraphs above) of
liver cells as well as of cells from other organs comprising a
mammalian organism, and of the various organs themselves. These
assays may be variously utilized for analytic, therapeutic,
diagnostic, or industrial purposes. However, at the current state
of the art, such in vitro cell-based assay systems possess
limitations that impose high costs, or that limit the simulative or
predictive capacities of the assay (which in turn imposes high
costs when the simulative or predictive results of the assay are
found to be of no or limited use). One form of limitation that
currently, frequently occurs in in vitro cell-based assay systems
is that the configuration of the system causes the degree of
metabolic or other functional competency of the cultured cells to
remain at too low a level to yield accurate, or measurable, results
that afford a useful prediction of how a chemical entity being
tested on the system will interact with a cell, an organ, an organ
system or an entire organism in vivo. Another challenge of
cell-based systems is that the system is configured such that a
level of cellular functionality, once achieved, cannot be
maintained over a desirable duration of time. Another such form of
limitation is that the amount of time that must be devoted to the
initial incubation and/or culture of the cellular materials, prior
to the time when the cells assume the higher or highest degrees of
functionality of which they become capable, is a time of long
duration. Improvements to the state of the relevant art, which may
serve to reduce any of these or other limitations or their impacts,
will provide more accurate and cost-effective means of using
cellular cultures.
SUMMARY OF THE INVENTION
Present herein is a system in which cells are maintained in a
metabolically highly active state from close to the onset of the
culture. The system may be utilized to test the potential
toxicological impacts of constituent components of consumer and
industrial products, to study the environmental impacts of
pollutants and other chemicals, to detect the presence of chemical
and bioweapons, to analyze the pharmacologic, pharmacokinetic and
toxicological properties of molecular entities, and to study other
interactions between cellular materials and chemical or molecular
entities.
From a commercial standpoint, certain cell types are not useful if
they exist in a low metabolic state. For example, testing drugs for
hepatotoxicity frequently cannot be adequately accomplished if the
hepatocytes are in a low metabolic state. Therefore, the period
during which the cells are in a low metabolic state constitutes a
period of low, no, or wasted productivity in economic terms.
Despite the absence or loss of productivity in the time during
which the low metabolic state obtains which may be as long as a
week or longer, such initial phase has been viewed and accepted by
industrial and academic scientists as an unavoidable prerequisite
that must be endured in order to reach at a later point of time a
culture characterized by a higher metabolic or other functional
state.
As presented herein there are embodiments in which the initial
phase of low metabolic state is reduced significantly to an extent
that useful applications of cultured cells are provided. In other
aspects, other embodiments are described in which other limitations
associated with traditional cell culture are reduced significantly
to an extent that useful applications of cultured cells are
provided.
Compositions and methods described herein utilize multiple cell
culture components. Each of the components of the system plays a
role distinct from the other components. Combinations of various
cell culture components give rise to various embodiments of
compositions and methods described herein. Also, each cell culture
component may have one or more embodiments. These cell culture
components combine together to provide unexpectedly rapidly arising
and high level of metabolic or other cellular function of the
cultured cells.
In one aspect, a cell culture component is a high oxygen
environment during cell seeding.
In one aspect, a cell culture component is a high oxygen
environment during cell culture.
In one aspect, a cell culture component is the absence of serum in
the culture media. Alternatively, said cell culture component is a
low level of serum in the culture media.
In one aspect, a cell culture component is the use of material that
enables the cells to assume a three-dimensional ("3D") relationship
to each other. In one embodiment, the cell culture component is a
three-dimensional scaffold. Alternatively, said cell culture
component is a gel sandwich, a gel overlay, a micropatterned array
of cells, cells configured in a spheroid, a tissue segment, a
tissue slice, or an artificial tissue construct.
In one aspect, a cell culture component is a cell cultured in the
presence of at least one additional cell type.
In one aspect, a cell culture component is liquid or gaseous cell
culture medium that is configured to contact cultured cellular
materials including cells cultured in a mono-layer, cells cultured
in a 3D relationship to each other, cells cultured in a co-culture,
subcellular materials, subcellular components, and cellular
products--under at least one condition of actuated perfusion or
flow.
Compositions and methods described herein comprise a cell culture
system comprising at least one compartment for culturing cells, a
culture of a first population of metabolically active cells and at
least two cell culture components selected from: (i) a cell culture
environment with an oxygen concentration higher than the
atmospheric concentration, (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
In one aspect of said cell culture system, said first population of
cells comprises hepatocytes.
In one aspect of said cell culture system, said structure for
three-dimensional culturing comprises a gel sandwich culture, a gel
overlay culture, a micropatterned overlay culture, a scaffold, a
tissue slice, a tissue segment culture, or an artificial tissue
construct.
In one aspect of said cell culture system, said second cell
population comprises non-parenchymal cells, stromal cells or immune
cells.
In one aspect of said cell culture system, said cell culture
environment comprises about 95% oxygen and about 5% CO.sub.2.
In one aspect of said cell culture system, said culture medium is
circulated to said at least one compartment under actuated flow or
perfusion through at least one microfluidic channel.
In one aspect, said cell culture system comprise a cell culture
environment comprising about 95% oxygen and about 5% CO.sub.2, a
three-dimensional culture comprising hepatocytes and fibroblasts
and a serum-free culture media. In another aspect, said cell
culture system further comprises a cell binding material on said
compartment for attachment of said cells. In another aspect, said
cell culture system further comprises at least one subcellular
component contained in the at least one compartment or another
separate compartment.
Compositions and methods described herein comprise a method of
culturing a first population of metabolically active cells
comprising culturing said cells in the presence of at least two
cell culture components selected from: (i) a cell culture
environment with an oxygen concentration higher than the
atmospheric concentration, (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
In one aspect of said method of culturing, said first population of
cells comprises hepatocytes. Alternatively, said first population
comprises kidney cells or keratinocytes.
In one aspect of said method of culturing, said oxygen
concentration is higher than atmospheric for the seeding of said
first population of cells.
In one aspect of said method of culturing, said structure for
three-dimensional culturing comprises a gel sandwich culture, a gel
overlay culture, a micropatterned overlay culture, a scaffold, a
tissue slice, a tissue segment culture, or an artificial tissue
construct.
In one aspect of said method of culturing, said second cell
population comprises fibroblasts, glial cells, endothelial cells,
stromal cells or non-parenchymal cells.
In one aspect of said method of culturing, said cell culture
environment comprises about 95% oxygen and about 5% CO.sub.2.
In one aspect of said method of culturing, primary cell hepatocytes
are seeded onto a three-dimensional cell culture structure together
with secondary cells comprising fibroblasts in an environment
comprising about 95% oxygen and about 5% CO.sub.2 and cultured in a
serum-free medium.
In one aspect of said method of culturing, said compartment further
comprises a coating of a binding material for attachment of said
cells.
Compositions and methods described herein comprise a method of
screening a material for its pharmacologic, metabolic,
pharmacokinetic, or toxicological properties comprising culturing a
first population of metabolically active cells in the presence of
at least two cell culture components selected from: (i) a cell
culture environment with an oxygen concentration higher than the
atmospheric concentration; (ii) a second cell population for
co-culture with said first population of cells and/or a structure
configured for three-dimensional culture of said first population
of cells with or without a second cell population for co-culture
with said first population of cells; (iii) a serum-free culture
medium or culture medium with a low concentration of serum; and
(iv) a cell culture medium that is configured to contact or come
into proximity with said first population of cells under at least
one condition of actuated perfusion or flow.
In one aspect of said method of screening, said method further
comprises measuring an activity of said first population of
cells.
In one aspect of said method of screening, said primary cells
comprise hepatocytes. Alternatively, said primary cells comprise
kidney cells or keratinocytes.
In one aspect of said method of screening, said activity of said
first cells comprises a metabolite, a biomarker, gene expression,
or protein activity.
In one aspect of said method of screening, said oxygen
concentration is higher than atmospheric for the seeding of said
primary cells.
In one aspect of said method of screening, said structure for
three-dimensional culturing comprises a gel sandwich culture, a gel
overlay culture, a micropatterned overlay culture, a scaffold, a
tissue slice, a tissue segment culture, or an artificial tissue
construct.
In one aspect of said method of screening, said method further
comprises contacting said first cells or the cell culture media
from said first cells with a subcellular component. In another
aspect of said method of screening, said method further comprises
measuring an activity of said subcellular component.
Compositions and methods described herein comprise a kit for
culturing cells comprising: (i) a device for culturing cells
containing at least one compartment, each compartment filled with
serum-free cell culture medium; (ii) a vial of a first population
of cryopreserved metabolically active cells; (iii) a vial of a
second population of cryopreserved cells; and (iv) a canister of
gases comprising about 95% oxygen and about 5% CO.sub.2.
In one aspect of said kit, said first population of cells comprise
hepatocytes.
In one aspect of said kit, said kit further comprises a scaffold
for three-dimensional cell growth at least one compartment.
In one aspect of said kit, said device comprises a microtiter
plate.
In one aspect of said kit, said device comprises a chip with at
least one microfluidic channel adapted to flow culture media
through said at least one compartment.
In one embodiment of compositions and methods described herein, the
system comprises multiple cell culture components.
In one embodiment of compositions and methods described herein, the
system employs three cell culture components.
In one embodiment of compositions and methods described herein, the
system employs two cell culture components.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in
this specification are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of compositions and methods described herein are
set forth with particularity in the appended claims. A better
understanding of the features and advantages of compositions and
methods described herein will be obtained by reference to the
following detailed description that sets forth illustrative
embodiments, in which the principles of compositions and methods
described herein are utilized, and the accompanying drawings of
which:
FIGS. 1 and 1A illustrates an embodiment of compositions and
methods described herein comprising multiple cell culture
components.
FIGS. 2 and 2A is illustrates an embodiment of compositions and
methods described herein comprising multiple cell culture
components.
FIGS. 3A and 3B illustrates effects of various cell culture
components on the metabolic state of cultured cells.
FIGS. 4A, 4B, and 4C illustrates effects of various cell culture
components on the metabolic state of cultured cells.
FIGS. 5A and 5B illustrates clearing of drugs by hepatocytes
cultured according to one embodiment of present invention.
FIG. 6 is a pro forma, prophetic graphical illustration of the
unexpected benefit which would be produced by an embodiment of
compositions and methods described herein comprising multiple cell
culture components.
DETAILED DESCRIPTION OF THE INVENTION
While preferred embodiments of compositions and methods described
herein have been shown and described herein, it will be obvious to
those skilled in the art that such embodiments are provided by way
of example only. Numerous variations, changes, and substitutions
will now occur to those skilled in the art without departing from
compositions and methods described herein. It should be understood
that various alternatives to the embodiments of compositions and
methods described herein may be employed in practicing compositions
and methods described herein. It is intended that the following
claims define the scope of compositions and methods described
herein and that methods and structures within the scope of these
claims and that their equivalents be covered thereby.
Described herein are configurations of elements of a cell culture
system for the purpose of producing, in an in vitro environment,
the enhanced cellular functionality of the cells cultured therein,
their subcellular components, cellular products, or cellular
materials, to an extent determined either in terms of at least one
of either (a) improved degree of superior functionality achieved or
(b) the improved rapidity with which any particular degree of
functionality is achieved than may be achievable by any alternative
cell culture system that does not comprise the component elements
that are configured in compositions and methods described herein.
Enhanced cellular functionality may comprise the cell's enhanced
ability to synthesize proteins or other cellular products; to
maintain the functionality of organelles or other subcellular
components, such as mitochondria; to produce cytokines; to express
genes; or to transport or metabolize xenogenous materials with
which the cell comes in contact; or to perform any other cellular
function Enhanced cellular functionality may comprise the degree,
or extent, to which the cell manifests any of the foregoing
functions.
Presented herein is a cell culture system in which cells rapidly
achieve and maintain a metabolically highly active state from close
to the onset of the culture. Various combinations of the cell
culture components described provide highly functional cells while
diminishing the time required to achieve such high functionality,
thus reducing time lost for productive use of the cultures.
From a commercial standpoint, certain cell types are not useful if
they exist in a low metabolic state, i.e., at a low level of
metabolic competency. For example, with respect to cultured
hepatocytes, testing for the hepatotoxicity or clearance of
chemical entities, or the generation of metabolites derived from
such entities, is not effective if the hepatocytes exist in a low
metabolic state. The period in which cells are in said low
metabolic state thus constitutes a period of low, or no, or wasted
productivity in economic terms. This limitation may diminish the
utility of culturing cells for a wide range of potentially
desirable uses including without limitation not only the study of
drug toxicity or drug metabolism, but also therapeutic uses (such
as a device comprising cultured cells that is used to treat
patients), diagnostic uses (such as, for example, a device
comprising cultured cells that is configured to measure some marker
in the blood of a patient), or industrial uses (such as, to cite
several examples, a device comprising cultured cells that is
configured to test for the potential toxicity of molecules that are
constituent elements of industrial or consumer products, or a
device comprising cultured cells that is configured to test for
levels of environmental pollutants or for the presence of chemical
or biological warfare agents). With the cell culture systems
described herein, this limitation is ameliorated and such uses as
those elaborated above become more effective, cost-effective, and
achievable. The embodiments presented herein contains various
combinations of components and conditions in which the initial
phase of low metabolic state is reduced providing quick achievement
of highly functional and metabolically active cell cultures useful
for a variety of purposes.
Described are multiple cell culture components. Each component of
the system is distinct from the other components and each
contributes to the unexpected benefits shown. Combinations of
various cell culture components give rise to different embodiments.
Also, each cell culture component may have one or more
embodiments.
In one aspect, a cell culture component employed in compositions
and methods described herein is a higher than normal atmospheric
oxygen environment. This may include high oxygen conditions for
seeding the cells and/or for the growth and culturing of the cells.
High oxygen conditions or environment comprise a concentration of
oxygen that is higher than a normally occurring, atmospheric
concentration of oxygen. In one embodiment, the concentration of
oxygen is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
or 100% of the gaseous composition present in the system. In
another embodiment, the concentration of oxygen is about 52%, 57%,
62%, 67%, 72%, 77%, 82%, 87%, 92%, or 97% of the total gaseous
composition present in the system. In some instances the
concentration of oxygen may be only slightly higher than the normal
atmospheric concentration. The oxygen concentration may be held
constant or relatively constant throughout the seeding or
culturing, or it may be changed over the course of the seeding or
culturing period.
There may be one or more non-essential gases present in the total
atmospheric composition utilized for high oxygen conditions. The
non-essential gas can be air, CO.sub.2, N.sub.2, or any type of
inert gas.
High oxygen seeding is accomplished by a number of approaches. For
example, high oxygen seeding is accomplished by seeding the cells
in an air-tight culture chamber. In an exemplary embodiment, cells
comprising a single cell type are seeded, i.e., plated, onto a
suitable physical substrate and then placed in an air-tight chamber
and the atmosphere is exchanged to high oxygen immediately
following the seeding. Alternatively, high oxygen seeding can be
accomplished by the use of a preconditioned chamber in which the
chamber already maintains a high level or concentration of
oxygen.
For high oxygen seeding, the duration in which oxygen level is
maintained higher than atmospheric level may vary from as little as
the time required for the seeding of the cell to as long as about
24 hours. In one embodiment, as soon as the seed culture is placed
in a cell culture chamber, oxygen level is returned to normal
atmospheric level. In another embodiment, the oxygen level is
maintained higher than atmospheric level for 24 hours.
Alternatively or in addition, high oxygen conditions may be
utilized after seeding. One could seed under normal oxygen
concentration and then increase the gaseous level of oxygen for the
period of time the cells are cultured. It is possible to utilize
high oxygen conditions for cell culture, for about 1, 2, 3, 4, 5,
6, 7, 8 or more days, or for any greater or lesser length of time.
In one embodiment, the high oxygen is used for seeding. In another
embodiment the high oxygen is utilized for the entire culturing
period. In another embodiment the high oxygen is utilized for a
portion of the culturing period. In another embodiment the high
oxygen is used intermittently.
For high oxygen conditions, the media can be treated by oxygen
bubbling. The high oxygen conditions in the closed chamber may be
accomplished just by utilizing oxygen bubbling through the medium.
Alternatively, the media can be used without the bubbling. In one
embodiment, the high oxygen content is achieved by increasing the
partial pressure of oxygen in the gaseous environment in the cell
culture device. It can be useful to begin with the high oxygen
content or to close the system including the cells and then alter
the oxygen content in the system. One may also enhance the oxygen
concentration of the media by the addition of an oxygen carrier,
such as hemoglobin or perfluorocarbon.
The level of oxygen can be monitored by various methods including
blood gas analyzer, monitoring atmospheric tension, oxygen monitor,
or other measuring devices known in the art. Monitoring of the
oxygen will permit one to maintain the conditions at the desired
oxygen level and for the period of time desired.
The oxygen level of the cell culture can be maintained by an
automated feed-back regulator type device. For example, a
computerized process can be employed to monitor the oxygen level
and adjust the input oxygen level accordingly. Such devices are
well known and available for this regulation.
In one aspect, a cell culture component is the absence of serum in
the culture media. Alternatively, said cell culture component is
low level of serum in the culture media. In one embodiment, the
cells are cultured in serum-free or substantially serum-free media.
A substantially serum-free medium might contain trace level of
serum. Serum-free media can be prepared or purchased from material
provided by serum-free media manufacturers. One example of such
media is Dulbecco's Modified Eagles Medium. Serum-free media may
include additives such as non-essential amino acids, antibiotics,
L-glutamine, or tryptophan. In another embodiment, the cells are
cultured in low-serum containing media. Examples of low serum
concentration level include about 0.1%, 0.2%, 0.5%, 0.7%, 1%, 1.2%,
1.5%, 1.7%, 2.0%, 2.2%, 2.5%, 2.7%, 3.0%, 3.2%, 3.5%, 3.7%, 4.0%,
4.2%, 4.5%, 4.7%, 5.0%, 5.2%, 5.5%, 5.7%, 6.0%, 6.2%, 6.5%, 6.7%,
7.0%, 7.2%, 7.5%, 7.7%, 8.0%, 8.2%, 8.5%, 8.7%, 9.0%, 9.2%, 9.5%,
9.7%, 10%, 10.2%, 10.5%, 10.7%, or 11%, or any intermediate value
between any of the forgoing. Alternatively, examples of low serum
level include about 11%, 11.5%, 12%, 13.5%, 14%, 14.5%, 15%, 15.5%,
16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% or any
intermediate value between any of the forgoing.
Exemplary sources of serum include, but not limited to, horse,
chicken, donkey, rabbit, cow, or rat.
In one aspect, a cell culture component is the use of material that
allows the cells to assume three-dimensional ("3D") relationships
to each other.
One approach for a 3D configuration comprises hepatocytes cultured
in a "gel sandwich" configuration, wherein matrices of collagen
fibers are configured both below and above the hepatocytes, and the
hepatocytes are cultured in between the layers. In a variant 3D
approach a monolayer of hepatocytes which is adhered to a rigid
coated or uncoated surface is overlaid with a layer of collagen
gel. A third 3D approach relies on the aggregation of hepatocytes
into spheroids following the seeding of the cells on a soft gel
such as Matrigel.TM. or a weakly adhering surface.
In an embodiment, the material that allows the cells to assume 3D
relationships is a three-dimensional scaffold. The
three-dimensional scaffold can be made with a biologically
non-toxic, inert material. The scaffold can be made with material
that allows cellular attachment. One example of such material is a
biocompatible gel formula made with calcium alginate which forms a
material for culturing the cells. Other examples of biological
coating materials include, but are not limited to, methylcellulose,
MATRIGELT.TM., BIOCOAT.TM., collagen, fibrinogen, fibronectin,
gelatin, laminin hyaluronin, hyluronic acid, or any of the family
of polyamines such as polylysines. Such materials encourage binding
and enhance culturing and also may provide a micro-scaffold in
which cells can multiply while maintaining three-dimensional
relationship to each other. In one embodiment, the scaffold is made
of a gel matrix. In another embodiment, the scaffold is
self-assembling peptide hydrogel. In one embodiment there is no
binding material on the scaffold. The cross-sectional dimensions of
the structural filaments or fibers of the scaffold may range from
20 microns to 1000 microns, although any size that accommodates the
cells, in the interstitial spaces of the scaffold, known as the
pores, will generally function. Pore sizes may range in size from 1
micron to 200 microns, or larger.
In an aspect, the culture device includes one or more chambers at
least one of which includes a physical structure containing a
material for the three-dimensional cell culture. Such structure may
be a microscale scaffold for the stable lodging of cellular
material.
In a variant approach, hepatocytes can be entrapped in microscale
scaffolds such as calcium alginate, inside which the hepatocytes
may assume a three-dimensional configuration with respect to each
other, such as a spheroid configuration. Yet other forms of 3D
hepatocyte culture are comprised of culturing a slice or a segment
of actual tissue drawn directly from the liver of a recently
deceased organism; or an artificial tissue construct. 3D cell
cultures may also comprise tissue slices or tissue segments drawn
from other organs, such as slices or segments of brain tissue, or
slices or segments of kidney tissue.
In one aspect, a cell culture component is a co-culture system. In
one embodiment, a co-culture system comprises a first cell
population and a second cell population wherein the second cell
population plays a supportive role in the metabolic or other
functionality of the first cell population. In an alternative
aspect, each cell population's presence in the co-culture system
serves to enhance the functionality of the other cell population
and of the co-culture as a whole. Examples of such a supportive
role include, but are not limited to, providing cytokines,
providing cell-cell contact, providing anchorage, excreting or
secreting extracellular material, and providing an environment
mimicking the in vivo environment of the first cell population. The
second cell population can be sub-lethally irradiated to prevent
the population from growing. The second cell population can be a
layer of cells attached to the planar surface of, or to a scaffold
configured inside, the chamber. The second cell population can be
configured to be cultured interspersed in or contiguous to the
first cell population. Alternatively, the second population can be
in suspension in the culture media. Exemplary sources of the second
cell population include stromal cells, non-parenchymal cells,
fibroblasts, glial cells, or immune cells. One can utilize various
immune cells, such as lymphocytes, dendritic or Kupffer cells. In
one embodiment, the second cell population is 3T3-J2 fibroblasts.
In one embodiment, the first and second cell populations are from
the same species of animal, such as human. In other cases, the cell
populations are from different species. It may be useful to obtain
the cells from any species of animal, including mammalian species
such as human, monkey, mouse, rat, pig, cow, horse, dog or sheep.
The cells may be freshly isolated cells, primary cells, engineered
cells, preserved cells, a cell line, a tissue slice, a tissue
segment, an artificial tissue construct, cryopreserved cells, or
stem cells.
In one embodiment, the co-culture system is comprised of two
different cell populations representing two different parenchymal
cell populations or representing a parenchymal cell population and
non-parenchymal cell population. For example, the first cell
population can be a kidney glomerular parietal cell population and
the second population can be a glomerular podocyte population. In
another example, the first cell population can be hepatocytes, and
the second population can be fibroblasts, endothelial cells, or
stellate cells. The proportion between the first cell population
and the second cell population can vary depending on the type of
cells. The ratio can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,
1:8, 1:9, 1:10, or vice versa, or any intermediate ratio between
any two of the foregoing, or any other ratio. In one embodiment,
hepatocytes are cultured with 3T3-J2 fibroblasts in a 10:1 ratio.
In another embodiment, hepatocytes are cultured with 3T3-J2
fibroblasts in a 2:1 ratio. In yet another embodiment, hepatocytes
are cultured with 3T3-J2 fibroblasts in a 1:1 ratio. Another
example is a gut epithelial population and a goblet cell
population. Another example is a Type I pneumocyte population and
an endothelial cell population. In another example the first
population is a keratinocyte population and the second population
is a Langerhan cell or dendritic cell population. In one
embodiment, the co-culture system is comprised of three or more
different cell populations representing parenchymal cell
populations, non-parenchymal cell populations, and immune cell
populations; for example, the first cell population can be
hepatocytes; the second population can be fibroblasts, endothelial
cells, or stellate cells; and the third population can be Kupffer
cells or T cells. Another example of a co-culture comprising three
or more different cell populations is a kidney glomerular parietal
cell population, a glomerular podocyte population, and an
endothelial cell population. Another such example is a co-culture
comprising a keratinocyte population, a fibroblast population, and
a Langerhan cell or dendritic cell population. Another such example
is a gut epithelial population, a goblet cell population, and a T
cell population. Another example is a Type I pneumocyte population,
a Type II pneumocyte population, and an endothelial cell
population. In one embodiment, members of the respective cell
populations are configured to be cultured in a particular geometric
arrangement and/or in particular proportions to one another by
means of a micropatterning technique.
A variety of configurations of the physical substrate for adhering,
holding or containing the cell culture system are possible,
including a flat substrate; or, in an embodiment, the physical
substrate may be configured to comprise at least one chamber or
compartment. The chamber comprises a planar surface and walls
surrounding and partially or completely enclosing the planar
surface. The planar surface is coated or treated to allow cellular
attachment. In an aspect, the chamber is open in one dimension,
thereby assuming the configuration of an open compartment or well.
In an alternative aspect, the chamber further comprises an
additional element configured to be continuously contiguous with
the walls of the chamber, so as to comprise a closed, air-tight
chamber suitable for the containment, without leakage, of gaseous
or liquid cell culture medium. The cell culture chamber is
structured to contain media as well as to withstand atmospheric
pressure accompanied with oxygenation. The chamber may or may not
comprise at least one opening through which sampling of the media
or atmosphere can be performed, or through which cell culture
medium can enter and/or exit the chamber, thereby perfusing any
cellular materials configured therein. In an embodiment, the cell
culture chamber is microscale, wherein microscale means being
configured to possess at least one physical feature that is
characterized by having at least one linear dimension (length,
width, height or depth) measuring less than one millimeter. Often
microscale is considered to be having dimensions of 10 nm to 1 mm
In an aspect, for ease and efficiency of use, the cell culture
chamber is repetitively configured with multiple embodiments
integrated proximally one to another into a single piece of
laboratory ware, such as a multi-well microtiter plate.
In one embodiment, a cell culture chamber can be used to
accommodate a monolayer cell culture. In an alternative embodiment,
the cell culture chamber is used to accommodate a cellular
co-culture, a culture of cells in a 3D configuration, subcellular
materials, cellular products, or subcellular components, any of
which may be primary, naturally occurring, man-made, artificial, or
engineered. The subcellular material in the culture device may be a
cellular product. Exemplary cellular products may include without
limitation an enzyme, a nucleic acid, a protein, a lipid, and a
carbohydrate. The cellular product may be man-made. The cellular
product may comprise a naturally occurring or man-made cellular
product in conjunction with some other biochemical entity. The
subcellular material may comprise a subcellular component.
Exemplary subcellular component may include without limitation a
microsome, mitochondrion, nucleus, ribosome, plasma membrane, and
the like. The subcellular component may be man-made. The
subcellular component may comprise a naturally occurring or
man-made subcellular component in conjunction with some other
biochemical entity. For example, the subcellular component may be
an engineered enzyme, protein or artificial cellular structure.
Such subcellular component may be included in any of the
compartments during the culturing step. In one embodiment, the
subcellular component is involved in the metabolic or toxicological
process of the system. For example, a material to be analyzed may
be cultured with the first population of metabolically active cells
to produce a product from the culturing step, which may interact
with the subcellular component. The result of that interaction may
be measured or analyzed to provide an evaluation of the
material.
In another embodiment the physical substrate for cell culture is a
biochip. The biochip comprises a microscale channel or channels
fluidically connected to or otherwise fluidically integrated with
at least one chamber or compartment for culture of cells or
subcellular materials. The at least one channel and the at least
one chamber are configured to facilitate the actuated, microfluidic
circulation or recirculation of cell culture medium in contact with
or in proximity to the cell culture system, under a condition of
perfusion or flow. In an aspect the configuration of the at least
one compartment and the at least one chamber comprises a linear
flow path for the circulation or recirculation of the cell culture
medium. In an alternative embodiment the flow path may bifurcate
and become multilinear. The biochip may be microscale. The at least
one compartment of the biochip may be configured to be either open
in one dimension, thereby assuming the configuration of an open
compartment or well; or closed so as to comprise a closed,
air-tight chamber suitable for the containment, without leakage, of
gaseous or liquid cell culture medium, as well as to withstand
atmospheric pressure accompanied with oxygenation. In an aspect,
the chamber also comprises an inlet and an outlet for flow of
culture medium.
An embodiment of the biochip may contain a single compartment
(e.g., a chamber); or alternatively, another embodiment of the
biochip may contain two compartments, where one compartment
contains cells, subcellular materials, subcellular components, or
cellular products and the other compartment is an open reservoir
for the addition or withdrawal of culture medium. In another
aspect, more than one chamber of the biochip may each contain cells
cultured in a monolayer, cells cultured in a co-culture,
subcellular materials, subcellular components, or cellular
products, wherein the cell culture system contained in one chamber
is different from the cell culture system contained in the at least
one other chamber. In an alternative embodiment, the cell culture
system in one compartment of the biochip is identical to the cell
culture system in the at least one other compartment of the
biochip. In another embodiment the biochip may contain at least one
compartment and in some instances three or more compartments.
Another embodiment of the biochip may further comprise a pumping
mechanism, wherein the pumping mechanism may either be integrated
in the biochip or separate from and external to the biochip. In one
such embodiment the pumping mechanism may be hydraulic, such as a
syringe pump or a peristaltic pump. In one such embodiment, the
pumping mechanism may be electro-kinetic or, alternatively, an
alternative embodiment may comprise a diaphragm pump that is
mechanically actuated or pneumatically actuated. In another
embodiment, the biochip may further comprise a de-bubbler located
within the biochip or external to the biochip. In another
embodiment the biochip may comprise at least one sensor for
obtaining signals from the cultured cells, subcellular materials,
subcellular components, or cellular products, wherein at least one
sensor may be a biosensor and the biosensor may comprise a
waveguide.
The biochip may be microfabricated. The biochip may be manufactured
from a microfabricated master. The biochip may be manufactured by
mass production that causes the geometry of the device (including
the provision for the rate of fluid flow in and through the device)
to be substantially the same from one such manufactured copy,
specimen or iteration of the device to the next. The process of
mass production may include that the biochip is manufactured from a
microfabricated master.
The well, chamber, microtiter plate, biochip, or other physical
substrate may be comprised of glass, silicon, a plastic such as
polycarbonate, cyclic oxide copolymer (COC), polystyrene, or other
plastic formulations, or any other material that may comprise a
suitable physical substrate for in vitro cell culture.
In one aspect, a cell culture component employed in compositions
and methods described herein is cell culture medium that is
configured to come into contact or proximity with a cell culture
system under a condition of perfusion or flow. The biochip, the
cell culture system and the pump may be configured together to
achieve desired or optimal levels of metabolic or other cellular
functions under the at least one condition of perfusion or flow.
The condition of perfusion or flow may be configured to maintain
desired values or ranges of certain parameters, such as the amount
of shear stress brought to bear upon the cells by the perfusate
culture medium, the flow volume or flow velocity of the perfusate
culture medium as it contacts or comes into proximity to the cell
culture system, the residence time during which a single molecule
of a chemical entity dissolved or suspended in the perfusate
culture medium remains within a compartment of the biochip or
remains in contact with the at least one cell culture system
contained in the biochip, and the like. Maintaining desires values
or ranges of these and other parameters may cause the cell culture
system to manifest desired or optimal levels of cellular function.
In one aspect, a desired value or range of at least one such
parameter simulates a value found in a living organism.
In an embodiment, the condition of perfusion or flow is maintained
during cell seeding. In an alternative embodiment, the condition of
perfusion or flow is maintained during cell culture. Parameters
that characterize the flow may be controlled so as to achieve
desired or optimal levels of cellular or subcellular functionality.
For example, in an embodiment the shear stress exerted by the
flowing cell culture medium upon the cell culture system is less
than 14 dynes per square centimeter (dyn/cm.sup.2). In an
alternative embodiment the shear stress exerted by the flowing cell
culture medium upon the cell culture system is less than 2
dyn/cm.sup.2. In other embodiments, the shear stress exerted by the
flowing cell culture medium upon the cell culture system and cells
is less than 1 dyn/cm.sup.2, less than 0.5 dyn/cm.sup.2, less than
0.2 dyn/cm.sup.2, less 0.1 dyn/cm.sup.2, less than one order of
magnitude less than 0.1 (0.01) dyn/cm.sup.2, less than two orders
of magnitude less than 0.1 (0.001) dyn/cm.sup.2, less than three
orders of magnitude less than 0.1 (0.0001), less than 0.00001, less
than 0.000001 dyn/cm.sup.2, less than 0.0000001 dyn/cm.sup.2, less
than 0.00000001 dyn/cm.sup.2, less than 0.000000001 dyn/cm.sup.2,
less than 0.0000000001 dyn/cm.sup.2. In another embodiment, the
cell culture system is configured in a biochip and the at least one
compartment of the biochip is in turn configured with a series of
ridges and depressions in its planar surface, such that the cell
co-culture is seeded down in the depressions of the compartment
while the ridges, which extend higher up into the flow path than
the cell culture system does, serve to mechanically shield the cell
culture system from the most forceful contacts with the perfusate,
such that the shear stress exerted upon the cell culture system is
minimal. In an alternative aspect, the compartment is configured to
comprise a permeable membrane that segregates the cell culture
system from the flowing perfusate and thereby shields it from shear
stress while permitting chemical entities dissolved or suspended in
the perfusate culture medium to come into contact and interact with
the cell culture system by diffusing across the membrane. In
another aspect, the flow rate of the perfusate cell culture medium
through the biochip is two nanoliters per minute (2 nL/min). In an
alternative aspect, the flow rate of the perfusate is five
microliters per minute (5 .mu.L/min) In a third exemplary aspect,
the flow rate is one milliliter per minute (1 mL/min) In another
embodiment, the flow rate is any intermediate value between any of
the foregoing rates. The flow rate may be held constant or, in an
alternative embodiment, it may vary or be intermittent during the
period of cell culture or during the period of cell seeding. In
another embodiment, the geometry of the biochip design and the
speed of the pumping mechanism are together configured to produce a
residence time in the at least one compartment of the biochip of
about 0.5 seconds (sec.), 0.75 sec., 1.0 sec., 5.0 sec., 20 sec.,
30 sec., 60 sec., 2 minutes (min), 5 min, 30 min, 60 min., 2 hours
(hr.), 3 hr., 4 hr., 6 hr., 12 hr., 24 hr., 48 hr., 96 hr., 1 week
(wk.), 2 wk., or any time value between any of the foregoing. In
one aspect, the residence time is configured to simulate at least
one residence time found in vivo in the human liver.
An example of an embodiment that comprises the multiple cell
culture components is high oxygen seeding in the presence of 95%
oxygen; cell culture media comprising a low concentration of serum;
non-parenchymal cells as the second cell population; and cell
culture medium that is configured to be pumped so as to
re-circulate through the compartment, where it comes into contact
with cells from at least one of the cell populations under at least
one condition of perfusion or flow. In another embodiment, an
example of the multiple cell culture components is high oxygen
seeding in the presence of 87.5% oxygen, cell culture medium
comprising no serum, non-parenchymal cells as the second cell
population, and cell culture medium that is configured to perfuse
the cell culture system in a biochip at a rate of 5 mL/min.
Alternatively, the cell culture system comprises multiple cell
culture components comprising high oxygen seeding in the presence
of 95% oxygen, cell culture media with 0.5% serum concentration,
non-irradiated fibroblasts as the second cell population, and a
three dimensional cell culture scaffold--including a layer of a
binding material, such as MATRIGEL.TM.. Another alternative example
of the multiple cell culture components comprises high oxygen
seeding in the presence of 100% oxygen, cell culture media with 1%
serum concentration, a co-culture comprising cryopreserved primary
human hepatocytes as the first cell population and 3T3-J2
fibroblasts as the second cell population, and flowing cell culture
medium that perfuses the co-culture (configured in a compartment of
a biochip) while maintaining a residence time in the compartment
similar to at least one value for residence time obtained in vivo
in the liver of an adult human, and while exerting in the
compartment a shear stress of less than 2 dyn./cm.sup.2. Another
embodiment includes high oxygen culturing in 95% oxygen, cell
culture media in serum free media, non-irradiated fibroblasts as
the second cell population, and a three dimensional cell culture
scaffold including a layer of a binding material, such as
MATRIGEL.TM..
In one embodiment, the cell culture system comprises multiple cell
culture components. One example of the multiple cell culture
components includes high oxygen seeding in the presence of 95%
oxygen, serum-free cell culture media, non-irradiated fibroblasts
as the second cell population, and a cell culture scaffold provided
by MATRIGEL.TM.. Another example of the multiple cell culture
components is high oxygen seeding in the presence of 100% oxygen,
serum-free cell culture media, 3T3-J2 fibroblasts as the second
cell population, and a cell culture scaffold provided by
MATRIGEL.TM.. Yet another example of the multiple cell culture
components is high oxygen seeding in the presence of 95% oxygen and
5% CO.sub.2, serum-free media, 3T3-J2 fibroblasts as the second
cell population, a cell culture scaffold provided by collagen
coating.
In another embodiment, the cell culture the system comprises three
cell culture components. One example of the three cell culture
components are high oxygen seeding in the presence of about 95%
oxygen, serum-free cell culture media, and non-irradiated
fibroblasts as the second cell population. Another example of the
three cell culture components are high oxygen seeding in the
presence of 100% oxygen, serum-free cell culture media, 3T3-J2
fibroblasts as the second cell population. Yet another example of
the three cell culture components are high oxygen culturing in the
presence of about 95% oxygen and about 5% CO.sub.2, serum-free cell
culture media, 3T3-J2 fibroblasts as the second cell
population.
In one embodiment, the cell culture the system comprises two cell
culture components. One example of the two cell culture components
is high oxygen seeding in the presence of 95% oxygen and cell
culture media with serum-free media. Another example of the two
cell culture components is high oxygen seeding in the presence of
100% oxygen and cell culture media with 0.1% serum. Yet another
example of the two cell culture components is high oxygen culturing
in the presence of about 95% oxygen and about 5% CO.sub.2 and
serum-free cell culture media.
It is possible to use any mammalian cell type, whether comprising a
primary cell or a cell line, for the cultures, including without
limitation hepatocytes, enterocytes, keratinocytes, neural cells,
cardiac muscle cells, pancreatic cells, renal cells, and stem
cells. Various embodiments of the culture system disclosed herein
can be customized as to cell type so as to obtain a high metabolic
or other functional state of the cells from close to the onset of
the culture; or to obtain at least one other unexpected benefit
from the culture. Compositions and methods described herein can be
useful for maintaining for an extended period of time primary cell
culture, i.e., cells freshly isolated from tissue or an organ; or
alternatively for maintaining for an extended period of time cell
lines, i.e., engineered cells adapted to grow and/or be maintained
and to function under in vitro conditions.
Primary cells can be obtained by any techniques known in the art.
Examples of such techniques include but are not limited to surgical
separation, isolation, fluorescence activated cell sorting,
magnetic activated cell sorting, use of a cell sieving device,
centrifugation, volume cell sorting, and chemotactic cell sorting
methods. In practice one may utilize multiple-parallel formats
wherein the format allows parallel culturing of cells under the
disclosed different combinations of cell culture components. In one
embodiment, the format is implemented in a micro-titer plate.
Examples of micro-titer plates include 6-well plate, 12-well plate,
24-well plate, 48-well plate, 96-well plate, 256-well plate,
384-well plate, and 1536-well plate.
Compositions and methods described herein can be manufactured as a
kit. In one embodiment a kit comprises a microtiter plate, oxygen
tank containing a mix of gases with pre-determined ratio of high
oxygen content, frozen vials containing one or more cell
population(s), and serum-free or low serum culture media. In one
embodiment a kit comprises a microtiter plate coated with a binding
agent and lyophilized cells and serum-free or low serum media. In
another embodiment the kit comprises a microfluidic chip including
channels for circulation of media to permit contacting the cells
under perfusion or flow, one or more compartments for culturing
cells, which compartments may include a variety of designs and
materials, such as biocompatible coatings of binding agents,
scaffolds for three-dimensional cell growth, reservoirs for fluids,
and ports for the injection of materials, gases, and monitoring of
oxygen or metabolic activity and products. Such chip may include
cells and media. Alternatively, the kits may include the chip,
vials of the primary and secondary cells and media for cell growth.
The kit may also include educational or training materials such as
an instruction manual or CD.
A variety of methods may be used to measure the metabolic,
toxicological, or other functional state of the cells. Methods of
measurement include any method to measure gene expression (e.g.
reverse transcription polymerized chain reaction ("RT-PCR")
analysis), any method to measure the expression level of enzymes or
other proteins produced in or by the cell (e.g. western blot or
ELISA), any method to measure the activity of enzymes or other
proteins produced by the cell (e.g. EROD
(ethoxyresorufin-O-deethylase), MROD
(methoxyresorufin-O-deethylase), PROD
(pentoxyresorufin-O-deethylase), BROD
(benzyloxyresorufin-O-deethylase) assays, or metabolite formation
by liquid chromatographic/mass spectroscopic analysis), any method
to measure active molecular transport into or out of the cell, any
method to measure the metabolic activity of organelles such as
mitochondria, any method to measure the production or secretion of
cytokines or chemokines, any method to measure the presence or
quantity of a biomarker indicative of a toxicological, signaling,
or other cellular process, or any other method used to measure an
aspect of cellular functionality. In one embodiment, hepatocytes
are cultured in a culture system comprising multiple cell culture
components and their metabolic states are measured by the level of
albumin. In another embodiment, hepatocytes are cultured in a
culture system having two, three or multiple components for three
days and their collective metabolic state is measured by reverse
transcription polymerized chain reaction ("RT-PCR") analysis. In
one embodiment, hepatocytes are cultured for a period of one, two,
three, or four days, or longer, in a culture system having two,
three or multiple components, in the presence of a potential
toxicant dissolved or suspended in the culture medium, and the
cells and the culture medium are subsequently assayed to determine
the presence of a biomarker indicative of the onset of an apoptotic
or otherwise cytotoxic process having incepted in the cell. In some
embodiments, the system may include five cell components--serum
free media, higher than atmospheric oxygenation, co-culture in a 3D
structural relationship and perfusion of the culture media. In one
embodiment, primary enterocytes or CACO-2 cells are cultured for a
period of one, two, or three days, or longer, in a culture system
having two, three or multiple components, in the presence of a
molecular entity dissolved and suspended in the culture medium,
wherein the primary enterocytes or CACO-2 cells are cultured on a
physical substrate that is configured with the enterocytes or
CACO-2 cells as a permeable membrane; and the cells and the culture
medium are subsequently assayed to determine the metabolic action
of CYP3A enzymes expressed in said cells upon said molecular
entity; and also to determine whether the cells have afforded the
absorption of the molecular entity through the permeable membrane.
In an embodiment, primary glomerular cells isolated from a human
kidney are cultured for a period of one, two, or three days, or
longer, in a culture system having two, three or multiple
components, in the presence of a molecular entity dissolved and
suspended in the culture medium, and the cells and the culture
medium are subsequently assayed to determine the presence of a
biomarker indicative of the onset of an apoptotic or otherwise
nephrotoxic process having incepted in the cell.
The devices and methods disclosed herein can be useful for drug
discovery and development or for consumer and industrial product
testing, or for environmental testing or biodefense applications.
Such testing includes, but is not limited to in vitro drug toxicity
testing and in vivo-surrogate testing. In one embodiment, the
culture systems described herein is used for the study of drug
clearance. In one embodiment it is used for the study of metabolite
generation. In one embodiment it is used for the study of
mechanisms of active or passive molecular transport. In one
embodiment it is used for the testing of the toxicity of substances
constituent to consumer products such as toothpaste, shampoo, hair
dye, or makeup. In one embodiment it is used for the testing of the
toxicity of substances constituent to industrial products such as
paint or insulation materials. In one embodiment it is used for
measuring the level of environmental pollutants such as dioxin. In
one embodiment, an environmental sample is extracted from water or
atmosphere and introduced into a culture medium, where it may
subsequently incite in the cell culture a response indicative of
toxicity, inflammation or hypersensitivity. In one embodiment it is
used for detecting the presence of a chemical or biological warfare
agent, such as aflatoxins. With the disclosed cell culture systems,
enhanced cellular function is manifested with respect to at least
one type of cellular function to produce a higher percentage of the
functionality that the cells would evidence in vivo, compared to
what those cells could produce under in vitro cell culture
conditions that were not so enhanced.
Alternatively, enhanced cellular functionality may comprise the
greater speed, or rapidity--i.e., the reduced time required--in
which cells may recover an equivalent degree of any one such type
of cellular functionality compared to the time in which those cells
could recover that same functionality under in vitro cell culture
conditions that were not so enhanced. The benefit of such
time-based enhanced cellular functionality is that it may increase
the aggregate amount of time during which the cell manifests the
full level of functionality it achieves in in vitro culture, or
diminish the time that elapses during the cell or tissue culture
process, before that high level of full in vitro functionality
incepts, thereby increasing the total time available, and
therefore, the total potential benefit derivable, from the cell
culture while in subsequent experimental use. In an alternative
aspect the benefit of such time-based, enhanced cellular
functionality is that it minimizes the time that is lost, and the
attendant labor and cost that is expended, compared to cell culture
methods that do not benefit from the enhanced functionality yielded
by compositions and methods described herein, which require longer
periods of time in culture.
The systems may be utilized to evaluate the toxicity, efficacy, and
pharmacokinetic disposition of new drugs more efficiently than is
currently possible because of the quicker achievement of a high
metabolic state by the cells. One aspect of the culturing systems
disclosed is the ability to detect the formation, accumulation
and/or further metabolic clearance of secondary metabolites as well
as the potential to elucidate the role of drug transporters in drug
clearance. For example, a parent molecular entity can be exposed to
the system and the culture medium can subsequently be repeatedly
sampled at predetermined time points; the samples can then by
analyzed by mass spectroscopy to investigate the prospective
formation and clearance of primary metabolites of the parent,
secondary metabolites of the parent, tertiary metabolites of the
parent, and subsequent generations of metabolites of the original
parent compound.
EXAMPLES
Exemplary Cell Culture System Comprising Multiple Cell Culture
Components
The system depicted in FIG. 1 comprises a compartment 102 with a
layer of binding material 103 configured to facilitate the physical
adherence of cellular materials to the physical substrate 101. The
binding material 103 is comprised of at least one material drawn
from the group consisting of MATRIGELT.TM., any of the cell culture
systems provided under the trade name BIOCOAT.TM., collagen,
fibrinogen, fibronectin, gelatin, laminin hyaluronin or hyaluronic
acid, or any of the family of polyamines such as polylysines. The
system 100 depicted in FIG. 1 also comprises a co-culture of at
least one type of metabolically active cell 104 located in
proximity with at least one type of non-parenchymal, stromal cell
105. The system 100 depicted in FIG. 1 also comprises a liquid cell
culture medium containing no serum 106 and a cell culture
environment 107 having an oxygen content higher than atmospheric
concentration.
The system depicted in FIG. 1A illustrates an alternative
embodiment of the system. Hepatocytes 104 and fibroblasts or other
types of stromal cells 105 are affixed in a particular geometry and
proximity to each other within a compartment 102 on the physical
substrate 101 in predetermined quantities and patterns using a
micropatterning technique. Binding material 103 is selectively
placed so as to cause the cells 104 and 105 to assume their desired
positions and geometry in the micropattern. The system 100 depicted
in FIG. 1A also shows a liquid cell culture medium containing no
serum 106 and a cell culture environment 107 having an oxygen
content higher than atmospheric concentration.
FIG. 2 illustrates an alternative embodiment of the system. The
system 100 depicted in FIG. 2 comprises protein layers 203A and
203B that are configured above and below the metabolically active
cells 204 that are configured in a monolayer between them. The
protein layers 203A and 203B and the metabolically active cells 204
collectively comprise a type of three-dimensional cell culture
configuration. Alternatively, the lower protein layer 203A may not
be not present while the upper protein layer 203B is configured to
be present in the system, comprising along with the cells 204 a
type of three-dimensional cell culture configuration. In yet
another embodiment of the system 100, the lower protein layer 203A
is not present but in its place is configured a layer of binding
material 103 within the compartment 102 of the substrate 101. Also
shown are the system 100, a liquid cell culture medium 106 and a
cell culture environment 107 having an oxygen content higher than
atmospheric concentration.
FIG. 2A illustrates an alternative embodiment of the system. The at
least one compartment 102 of the physical substrate 101 contains a
material that is configured to comprise a type of three-dimensional
cell culture microscale scaffold 301 for the stable lodging and/or
adherence of cellular material. The scaffold 301 may be comprised
of calcium alginate. The cross-sectional dimensions of the scaffold
301 may vary in size. A protein binding material 303 is configured
to affix and stabilize points of the scaffold 301 to the
compartment 102. Alternatively, protein binding material 303 may be
absent. Metabolically active cells 304 are configured to be lodged
and stabilized in the pores of the scaffold, agglomerating into
non-linear configurations of multiple cells such as (but not
limited to) spheroids. Also shown are the system 100, a liquid cell
culture medium 106 and a cell culture environment 107 having an
oxygen content higher than atmospheric concentration.
Effect of Various Cell Culture Components on the Metabolic State of
Cultured Cells
In FIGS. 3A and B, freshly isolated hepatocytes are co-cultured
with 3T3-J2 fibroblasts, in the presence of serum-containing media
and atmospheric oxygen. Albumin production slowly increases over
time stabilizing at 80 .mu.g/1.times.10.sup.6 cells/24 hrs
following 11 days of co-culture. Gene expression analysis of Phase
I and Phase II enzymes such as CYP450 enzymes in the case of Phase
I enzymes, or glucuronidation enzymes or sulfation enzymes in the
case of Phase II enzymes], as well as transporter proteins is
carried out by quantitative reverse transcription polymerase chain
reaction (qRT-PCR). Gene expression at the onset of culture is
minimal and is significantly lower than in vivo levels of
transcription.
In FIG. 4A, the gene expression of hepatocytes co-cultured with
non-parenchymal cells in serum free media, and under high oxygen
tensions (95% O.sub.2, 5% CO.sub.2) at the first day of culture
(Day 1), is compared to that of fresh hepatocytes. Gene expression
analysis of phase I and phase II enzymes, as well as transporter
proteins is carried out by quantitative reverse transcription
polymerase chain reaction (qRT-PCR). Gene expression at the onset
of culture is comparable to in vivo levels of gene
transcription.
In FIG. 4B, long-term synthetic function of oxygenated cultures is
tested. In FIG. 4C, long-term cyp1A1/2 activity is measured. During
the first day of culture, albumin secretion is 4-fold higher in
oxygenated, serum-free, co-cultures compared to hepatocytes
co-culture with 3T3-J2 fibroblasts under standard conditions
(P=0.011 N=3) demonstrating the more rapid achievement of metabolic
function. No significant difference is detected at day seven of
culture. To compare long-term cyp1A1/2 activity to hepatocytes in
suspension, freshly isolated hepatocytes as described above is
compared to isolated hepatocytes following 2 hours of incubation at
37.degree. C. to control for the rapid loss of function in
suspension. FIG. 4C shows that cyp1A1/2 activity in oxygenated
cultures is comparable to that of hepatocytes in suspension. During
the first day of culture, the activity is 135% higher than that of
hepatocytes co-cultured with 3T3-J2 under standard conditions
(P=0.001 N=3). However, at day 7 of culture, there is no
significant difference between the cultures, demonstrating a
benefit of the combinations.
Clearing of Drugs in Hepatocytes Cultured According to One
Embodiment
Cryopreserved human hepatocytes are cultured with 3T3-J2
fibroblasts under serum free conditions. Following overnight
incubation in high oxygen tension (95% O.sub.2, 5% CO.sub.2) the
cells are exposed to one of the following drugs: antipyrine,
buspirone, metoprolol, sildenafil, and timolol. FIG. 5A shows the
clearance of all drugs including slow clearing drugs such as
antipyrine, sildenofil, and timolol which could not be assayed
using suspension cultures. FIG. 5B shows the long term accumulation
of 4-OH midazolam in our cultures in the presence and absence of
rifampicin an inhibitor of the drug transporter Oatp-2. The
accumulation of 4-OH midazolam in the culture media can be detected
over 30 hours of culture. This allows the completion of long term
experiments and evaluations during the first few days of
culture.
High Metabolic State of Hepatocyte at the Onset of Cell Culture
High metabolic state may comprise the cell's enhanced ability to
synthesize proteins or other cellular products; to maintain the
functionality of organelles or other subcellular components, such
as mitochondria; to produce cytokines; to express genes; or to
transport or metabolize xenogenous materials with which the cell
comes in contact; or to perform any other cellular function High
metabolic state may comprise the degree, or extent, to which the
cell manifests any of the foregoing functions. High metabolic state
may therefore be manifested by cell culture systems the unique
configurations of which endow their constituent cells with the
ability with respect to at least any one type of cellular function
to produce a higher percentage of the functionality that they would
evidence in vivo, compared to what those cells could produce under
in vitro cell culture conditions that were not so enhanced.
Table 1 illustrates the advantages of the transition to the
components of compositions and methods described herein comprised
by serum-free (or alternatively, low serum concentration) media
formulation, higher than atmospheric oxygen concentration, or both.
In this experimental configuration hepatocytes are cultured alone
or co-cultured with non-parenchymal cells, in the presence or
absence of serum. Two seeding conditions were evaluated: one was
normal atmospheric condition and the second was 95% oxygen, both
under 5% CO.sub.2 as is common practice. Following overnight
seeding, the cultures were washed and the level of demonstrated
cellular functionality at first day of culture, as exemplified by
CYP1A1/2 activity, was measured by the ethoxyresorufin-O-deethylase
(EROD) assay under normal oxygen condition. The result shows that,
compared to the baseline condition of monocultured hepatocytes,
each respective embodiment of compositions and methods described
herein confers an improved cellular functionality at first day of
culture. It should be emphasized that the data in Table 1 is not
simply the attainment of a higher level of cellular function, but
the attainment of that higher level very rapidly, at the first day
of culture, thereby enabling the culture to be productively
utilized on as many as six, nine, or twelve or more additional,
earlier days than would be afforded under traditional cell culture
or co-culture methods.
TABLE-US-00001 TABLE 1 CYP1A1/2 activity at first day of culture
demonstrated by various embodiments of compositions and methods
described herein Cell culture components comprised in various
embodiments: none serum- (hepatocyte co- high free CYP1A1/2
activity monoculture) culture oxygen media (nM/min/1 .times.
10.sup.6 cells) X 0.89 .+-. 0.20 X X 1.45 .+-. 0.35 X X 2.44 .+-.
0.20 X X 2.63 .+-. 0.13 X X X 3.45 .+-. 0.21
Table 2 compares the metabolic function of hepatocytes co-cultured
with non-parenchymal cells in serum free media, and under high
oxygen tensions (95% O.sub.2, 5% CO.sub.2) at the first day of
culture (Day 1), to that of freshly isolated hepatocytes cultured
in suspension (Day 0). The activities of CYP1A1/2 and CYP2B1/2 are
measured using the EROD, MROD, PROD and BROD assays respectively.
As can be seen, the metabolic activity of hepatocytes in oxygenated
co-cultures is equivalent or higher than that of hepatocytes in
suspension.
TABLE-US-00002 TABLE 2 Cyp1a1/2 and Cyp2b1/2 activity (nM/min/1
.times. 10.sup.6 cells) EROD MROD PROD BROD Suspension 2.73 .+-.
0.07 0.74 .+-. 0.10 0.24 .+-. 0.03 0.04 .+-. 0.08 (Day 0)
Oxygenated co- 3.10 .+-. 0.40 0.83 .+-. 0.10 0.85 .+-. 0.35 0.23
.+-. 0.07 culture (Day 1)
FIG. 6 illustrates, on a pro forma, prophetic, conceptual basis,
the time-based benefit derived from an embodiment of a cell culture
system described herein, wherein the benefit area is depicted in
the Figure as the cross-hatched area. In FIG. 6, the low bell curve
that shows cellular functionality enduring at a low level from 0-10
days represents the performance of plated cryopreserved primary
hepatocytes cultured in a mono-culture under traditional culture
methods and conditions. The second bell curve that shows cellular
functionality rising to a relatively higher level by Day 14
represents the performance of a traditional culture of a co-culture
of plated cryopreserved primary hepatocytes with 3T3 fibroblasts.
This represents co-culture without any at least a second cell
culture component. The large bell curve that encompasses both the
cross-hatched area and the area bounded by the second bell curve
reflects the level of cellular function envisioned by at least one
embodiment, including materially more rapid attainment of high
functionality very close to the time of onset of the cell culture.
The high cellular function beginning on the first day after culture
permits use of the cell cultures more quickly than in the past;
reduces tissue culture, labor, and inventory carrying costs;
creates additional days of high cellular function during which the
cell culture can be utilized for economically productive purposes,
which days would otherwise have to be devoted to the economically
unproductive, prerequisite task of additional tissue incubation and
culture; and provides a more efficient system.
A Multi-Well Format Embodiment of Compositions and Methods
Described Herein
A 96-well plate is coated with hydrogel. Each well is filled by
serum-free media. 3T3-J2 fibroblasts are placed to each well and
the plate is cultured overnight. On the following day, human
hepatocytes are thawed from a cryopreserved vial and placed in 95%
oxygen chamber with serum-free media. The cells are counted. To
each well, discrete, pre-determined numbers of cells are seeded
while maintaining high oxygen level. The 96-well plate is then
culture overnight in the presence of 95% oxygen. The plate is
subsequently moved to a cell culture incubator with normal oxygen
level. 12 different drugs are diluted in serum free media and then
added to the first row of 96-well plate. The addition is repeated
in subsequent rows of 96-well plate. 5 days later, MROD assays are
conducted with cell extracts obtained from each well.
In an alternative multi-well embodiment, the cell culture system is
comprised in one compartment of a multi-well assay platform that is
configured to comprise a culture of artificially or naturally
occurring cellular material in at least one additional compartment
of the platform. The several compartments of the assay platform are
microfluidically interconnected by at least one microscale channel
configured to conduct liquid or gaseous cell culture medium in a
circulating or re-circulating pathway between and among the
compartments.
In an alternative embodiment, the cell culture system is configured
to comprise a cell culture compartment that is microfluidically
connected to at least one microscale input or output channel,
wherein the channel and the compartment are configured to conduct
liquid or gaseous cell culture medium in a circulating or
re-circulating pathway, and wherein the cell culture medium being
configured to come into contact with the cell culture comprising
compositions and methods described herein under at least one
condition of perfusion or flow comprises an additional, separate
and distinct component of compositions and methods described
herein.
Kits
A kit is manufactured including the following components:
microtiter plate, oxygen tank containing mix of gases with
pre-determined ratio, an instruction manual, an instruction compact
disc, frozen vials containing second cell population, and culture
media, which may be low serum or serum free media. Alternatively, a
kit is prepared containing a microtiter plate coated with a binding
agent layer or a material to form a microscaffold and containing
serum-free or low serum media. Included are vials of cryopreserved
primary and secondary cells. In another embodiment, the kit
comprises a chip including at least one compartment connected with
microfluidic channels to permit perfusion of the culture media,
oxygen containers with a mixture of gases to provide a higher than
atmospheric concentration of oxygen, and a serum free culture
media.
* * * * *